Copper (Cu) is recognized as an essential trace elements for the body; However, excessive levels of Cu can lead to toxic effects. We investigated the effects of Cu2+(75 μg/L, 150 μg/L, and 300 μg/L) on the rainbow trout liver. Combination of transcriptome and metabolome analyses, the regulatory mechanisms of the liver under Cu stress were elucidated. The results showed that Cu affected the antioxidant levels, leading to disruptions in the normal tissue structure of the liver. Combined transcriptome and metabolome analyses revealed significant enrichment of the insulin signaling pathway and the adipocytokine signaling pathway. Additionally, Cu2+ stress altered the amino acid metabolism in rainbow trout by reducing serine and arginine levels while increasing proline content. Apoptosis is inhibited and autophagy and lipid metabolism are suppressed; In summary, Cu2+ stress affects energy and lipid metabolism, and the reduction of serine and arginine represents a decrease in the antioxidant capacity, whereas the increase in proline and the promotion of apoptosis potentially serving as crucial strategies for Cu2+ resistance in rainbow trout. These findings provided insights into the regulatory mechanisms of rainbow trout under Cu2+ stress and informed the prevention of heavy metal pollution and the selection of biomarkers under Cu pollution.

Infectious pancreatic necrosis virus (IPNV) is an important pathogen that is threatening the worldwide salmon and trout industry. But there is no therapeutic drug available for now. In this study, we demonstrate that MK-0608 is highly efficient against IPNV and low cytotoxic, with a 50 % effective concentration (EC50) of 0.20 μM and selectivity index (SI) of about 268. Time of addition assay illustrated that MK-0608 targeted the early stage of IPNV life cycle. Furthermore, we found that MK-0608 blocked IPNV attachment on the premise of sufficient pre-incubation time but MK-0608 did not influence viral internalization and release. MK-0608 could inhibit IPNV genome synthesis, and combination with ribavirin enhanced the inhibition effect, which might be functional via binding to IPNV RNA dependent RNA polymerase (RdRp), which was predicted by using molecular docking methods. In vivo test showed that IPNV was extremely suppressed in the rainbow trout (Oncorhynchus mykiss) with one single dose of MK-0608, and the higher dosage of 50 mg/kg could cause 3 log decrease of IPNV loads in fish tissues.

Rainbow trout (Oncorhynchus mykiss) is an economically significant freshwater-farmed fish worldwide, and the frequent outbreaks of infectious hematopoietic necrosis (IHN) in recent years have gravely compromised the healthy growth of the rainbow trout aquaculture industry. Fish skin is an essential immune barrier against the invasion of external pathogens, but it is poorly known about the role of circRNAs in rainbow trout skin. Therefore, we examined the expression profiles of circRNAs in rainbow trout skin following IHNV infection using RNA-seq. A total of 6607 circRNAs were identified, of which 34 circRNAs were differentially expressed (DE) and these DE circRNA source genes were related to immune-related pathways such as Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, Cytokine-cytokine receptor interaction, ubiquitin mediated proteolysis, and ferroptosis. We used qRT-PCR, Sanger sequencing, and subcellular localization to validate the chosen DE circRNAs, confirming their localization and expression patterns in rainbow trout skin. Further, 12 DE circRNAs were selected to construct the circRNA-miRNA-mRNA regulatory network, finding one miRNA could connect one or more circRNAs and mRNAs, and some miRNAs were reported to be associated with antiviral immunity. The functional prediction findings revealed that novel_circ_002779 and novel_circ_004118 may act as sponges for miR-205-z and miR-155-y to regulate the expression of target genes TLR8 and PIK3R1, respectively, and participated in the antiviral immune responses in rainbow trout. These results shed light on the immunological mechanism of circRNAs in rainbow trout skin and offer fundamental information for further research on the innate immune system and breeding rainbow trout resistant to disease.

Long non-coding RNA (lncRNA) is a novel emerging type of competitive endogenous RNA (ceRNA) that performs key functions in multiple biological processes. However, little is known about the roles of lncRNA under hypoxia stress in fish. Here, vascular endothelial growth factor-Aa (vegfaa) was cloned in rainbow trout (Oncorhynchus mykiss), with the complete cDNA sequence of 2914 bp, encoding 218 amino acids. The molecular weight of the protein was approximately 25.33 kDa, and contained PDGF and VEGF_C domains. Time-course and spatial expression patterns revealed that LOC110520012 was a key regulator of rainbow trout in response to hypoxia stress, and LOC110520012, miR-206-y and vegfaa exhibited a ceRNA regulatory relationship in liver, gill, muscle and rainbow trout liver cells treated with acute hypoxia. Subsequently, the targeting relationship of LOC110520012 and vegfaa with miR-206-y was confirmed by dual-luciferase reporter analysis, and overexpression of LOC110520012 mediated the inhibition of miR-206-y expression in rainbow trout liver cells, while the opposite results were obtained after LOC110520012 silencing with siRNA. We also proved that vegfaa was a target of miR-206-y in vitro and in vivo, and the vegfaa expression and anti-proliferative effect on rainbow trout liver cells regulated by miR-206-y mimics could be reversed by LOC110520012. These results suggested that LOC110520012 can positively regulate vegfaa expression by sponging miR-206-y under hypoxia stress in rainbow trout, which facilitate in-depth understanding of the molecular mechanisms of fish adaptation and tolerance to hypoxia.

Rainbow trout is a widely farmed economical cold-water fish worldwide, but the prevalence of infectious hematopoietic necrosis virus (IHNV) presents a severe risk to the aquaculture industry, resulting in high mortality and huge economic losses. In this study, the impacts of different concentrations (0, 10, 20, and 30 g/kg) of Chinese herbal medicine mixture (CHMM) on the immune response and resistance of rainbow trout to IHNV infection were evaluated. The results show that CHMM noticeably increased (P < 0.05) T-SOD, CAT, AST, ALT, ACP, and AKP activities and decreased MDA content. NF-κB, TNF-α, IFN-β, IL-1β, JAK1, HSP70, and HSP90 expressions were significantly upregulated (P < 0.05) in all CHMMs, while SOCS2 expression was downregulated (P < 0.05). Following infection with IHNV, feeding rainbow trout with varying amounts of CHMM resulted in noticeably increased (P < 0.05) T-SOD, ACP, and AKP activities and significantly decreased (P < 0.05) MDA content and AST and ALT activities. TNF-α, IFN-β, IL-1β, HSP70, and HSP90 expressions were significantly upregulated (P < 0.05) in all CHMMs, while the expressions of JAK1 and SOCS2 were downregulated. The expression level of the IHNV G protein gene at a dosage of 20 g/kg was notably lower than that of the other CHMM feeding groups. This study provides a solid scientific basis for promoting CHMM as an immunostimulant for boosting antiviral immunity in rainbow trout.

Hypoxia, largely triggered by global warming and water contamination, has become an environmental issue of great concern, posing a great threat to aquatic ecosystem. As one of the world\'s most economically important fish, rainbow trout (Oncorhynchus mykiss) is extremely intolerant of hypoxic environments, however, little is known about the roles of non-coding RNAs (ncRNAs) in the response of rainbow trout to hypoxia stress. Herein, effects of moderate (Tm12L) and severe hypoxia for 12 h (Ts12L) and 12 h reoxygenation on histology, biochemical parameters (antioxidant, metabolism and immunity) and transcriptome (lncRNA, miRNA and mRNA) in rainbow trout liver were investigated. We further validated the regulatory relationships between LOC110519952, novel-m0023-5p and glut1a via dual‑luciferase reporter, overexpression and silencing assays. Compared with Tm12L, the liver in Ts12L showed more severe oxidative damage. Anaerobic, lipid and protein metabolism was enhanced under hypoxia stress, especially in Ts12L. We also found that Tm12L could strengthen innate immune response, which was inhibited in Ts12L. Besides, several hypoxia-related genes (glut1a, vegfaa, hmox, epoa, foxo1a and igfbp1) and ceRNA networks were identified from 1824, 427 and 545 differentially expressed mRNAs, miRNAs and lncRNAs, including LOC118965299-novel-m0179-3p-epoa, LOC110519952-novel-m0023-5p-glut1a, MSTRG.7382.2-miR-184-y-hmox and LOC110520012-miR-206-y-vegfaa. Through in vitro and in vivo functional analysis, we demonstrated that glut1a is a target of novel-m0023-5p, and LOC110519952 can positively regulate glut1a by targeting novel-m0023-5p. Introduction of LOC110519952 could attenuate the promoting effects of novel-m0023-5p on rainbow trout liver cell viability and proliferation. This study highlights the differences in the regulatory mechanism of rainbow trout under different concentrations of hypoxia stress and provides valuable data for further research on the molecular mechanisms of fish adaptation to hypoxic environments.

In this study, the impact of three unsaturated fatty acids (Oleic acid: OA, Eicosapentaenoic acid: EPA, Docosahexaenoic acid: DHA) on the oxidation and structure of rainbow trout myofibrillar protein (MP) was explored. The findings revealed a notable increase in carbonyl content (P < 0.05) and a significant decrease in total sulfhydryl content (P < 0.05) of MP with the concentration increase of the three unsaturated fatty acids. Endogenous fluorescence spectroscopy and surface hydrophobicity analyses showed that unsaturated fatty acids can cause unfolding and exposure of hydrophobic groups in MP. In addition, SDS-PAGE showed that disulfide bonds were associated with MP cross-linking and aggregate size induced by unsaturated fatty acids. Overall, three unsaturated fatty acid treatments facilitated the oxidation of myofibrillar proteins, and the extent of protein oxidation was closely associated with the concentration of unsaturated fatty acids.

Infectious hematopoietic necrosis (IHN), caused by IHN virus, is a highly contagious and lethal disease that seriously hampers the development of rainbow trout (Oncorhynchus mykiss) aquaculture. However, the immune response mechanism of rainbow trout underlying IHNV infection remains largely unknown. MicroRNAs act as post-transcriptional regulators of gene expression and perform a crucial role in fish immune response. Herein, the regulatory mechanism and function of miR-206 in rainbow trout resistance to IHNV were investigated by overexpression and silencing. The expression analysis showed that miR-206 and its potential target receptor-interacting serine/threonine-protein kinase 2 (RIP2) exhibited significant time-dependent changes in headkidney, spleen and rainbow trout primary liver cells infected with IHNV and their expression displayed a negative correlation. In vitro, the interaction between miR-206 and RIP2 was verified by luciferase reporter assay, and miR-206 silencing in rainbow trout primary liver cells markedly increased RIP2 and interferon (IFN) expression but significantly decreased IHNV copies, and opposite results were obtained after miR-206 overexpression or RIP2 knockdown. In vivo, overexpressed miR-206 with agomiR resulted in a decrease in the expression of RIP2 and IFN in liver, headkidney and spleen. This study revealed the key role of miR-206 in anti-IHNV, which provided potential for anti-viral drug screening in rainbow trout.

Chemokines are cytokines with chemoattractant capacities that exert their physiological functions through the binding of chemokine receptors. Thus, chemokine and receptor complexes exert important roles in regulating development and homeostasis during routine immune surveillance and inflammation. Compared to mammals, the physiology and structure of chemokine receptors in fish have not been systematically studied. Furthermore, the salmonid-specific whole genome duplication has significantly increased the number of functional paralogs of chemokine receptors. In this context, in the current study, trout exhibited 17 cxcr genes, including 12 newly identified and 5 previously identified receptors. Interestingly, gene expression of brain cxcr1 and cxcr4, kidney cxcr3 and cxcr4, and spleen cxcr3, cxcr4, and cxcr5 subtypes were altered by bacterial infection, whereas brain cxcr1, kidney cxcr1 and cxcr7, and liver cxcr2, cxcr3, and cxcr4 subtypes were changed in response to environmental changes. Based on protein structures predicted by ColabFold, the conserved amino acids in binding pockets between trout CXCR4.1 subtypes and human CXCR4 were also analyzed. Our study is valuable from a comparative point of view, providing new insights into the identification and physiology of salmonid chemokine receptors.

Fibroblast growth factor 1 (FGF1) regulates vertebrate cell growth, proliferation and differentiation, and energy metabolism. In this study, we cloned rainbow trout (Oncorhynchus mykiss) fgf1 and fgf1a, prepared their recombinant proteins (rFGF1 and rFGF1a), and described the molecular mechanisms by which they improve glycolipid metabolism in carnivorous fish. A 31-d feeding trial was conducted to investigate whether they could enhance glycolipid metabolism in rainbow trout on high-carbohydrate diets (HCD). A total of 720 rainbow trout (8.9 ± 0.5 g) were equally divided into 4 groups: the chow diet (CD) group injected with PBS, the HCD group injected with PBS, the HCD group injected with rFGF1 (400 ng/g body weight), and the HCD group injected with rFGF1a (400 ng/g body weight). The results showed that short-term HCD had a significant positive effect on the specific growth rate (SGR) of rainbow trout (P < 0.05). However, it led to an increase in crude fat, serum triglyceride (TG) and glucose content, as well as serum glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) contents (P < 0.05), suggesting a negative health effect of HCD. Nevertheless, rFGF1 and rFGF1a showed beneficial therapeutic effects. They significantly reduced the crude fat content of the liver, serum TG, GOT, and GPT contents caused by HCD (P < 0.05). The upregulation in atgl, hsl, and acc2 mRNAs implied the promotion of TG catabolism. Moreover, rFGF1 and rFGF1a contributed to promoting lipolysis by activating the AMPK pathway and reducing lipid accumulation in the liver caused by HCD. In addition, the rFGF1 and rFGF1a-treated groups significantly reduced serum glucose levels and elevated hepatic glycogen content under HCD, and increased glucose uptake by hepatocytes. We observed a decrease in mRNA levels for pepck, g6pase, and pygl, along with an increase in mRNA levels for gys, glut2, and gk in the liver. Furthermore, these proteins regulated hepatic gluconeogenesis and glycogen synthesis by increasing the phosphorylation level of AKT, ultimately leading to an increase in GSK3β phosphorylation. In conclusion, this study demonstrates that rFGF1 and rFGF1a can enhance lipolysis and glucose utilization in rainbow trout by activating the AMPK pathway and AKT/GSK3β axis.